The underlying goal of this research program is to understand the mechanism of regulation of biological activity in macromolecular complexes through structural analysis. We use X-ray crystallography as a tool to achieve this objective. 1. The structural biology of complexes of 14-3-3. 14-3-3 proteins are a highly conserved family of homo- and heterodimeric proteins found within all eukaryotic cells. An important common characteristic is the ability of 14-3-3 proteins to bind other proteins in a phosphorylation dependent manner. Biological roles of 14-3-3 complexes have been demonstrated in signal transduction, subcellular targeting, and cell cycle control. During the past year, we have focused on the complex between 14-3-3 and Foxo4 (AFX), a transcription factor that functions in the insulin signaling pathway. We have performed biophysical characterization of the interactions between 14-3-3 zeta, Foxo4 and double stranded DNA representing part of an insulin response element. Analytic gel filtration and sedimentation equilibrium revealed that, upon PKB-mediated phosphorylation, Thr28 and Ser193 are binding sites for 14-3-3. Furthermore, doubly phosphorylated Foxo4 (11-213) binds 14-3-3 with significantly higher affinity than singly phosphorylated mutants. Structural characterization is in progress. 2. Adeno-associated virus (AAV) Rep mediates site-specific chromosomal integration. AAV has the unique ability to site-specifically integrate its genome into human chromosome 19, a property which places it in the forefront of much current interest as a vector for gene therapy. For instance, an AAV based vector system has been used successfully to incorporate a synthetic insulin gene into diabetic rats and mice, leading to remission of the disease state (Lee et al. 2000, Nature 408, 483). The viral DNA encodes for the Rep protein which possesses a site-specific DNA binding activity, an endonuclease activity, an ATPase activity, and a 3'-to-5' helicase activity. The structural basis of these activities is currently unknown. To shed light onto the mechanism of integration, we have determined the structure of the nuclease domain of AAV5 Rep protein at 1.4 Angstroms resolution. The domain displays a novel endonuclease fold and defines the three dimensional structural features of the rolling circle replication superfamily of proteins. Unexpectedly the structure shows three dimensional homology to other viral origin binding proteins such as the papillomavirus E1 protein and SV40 T antigen.